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LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis

Anders, Emma LU ; Dahl, Sara LU orcid ; Svensson, Daniel LU and Nilsson, Bengt Olof LU orcid (2018) In Biochemical and Biophysical Research Communications 501(1). p.280-285
Abstract

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a... (More)

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.

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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antimicrobial peptide (AMP), Cathelicidin, Clathrin, Host defense peptide (HDP), Innate immunity, Osteoblast
in
Biochemical and Biophysical Research Communications
volume
501
issue
1
pages
6 pages
publisher
Elsevier
external identifiers
  • scopus:85046791818
  • pmid:29729269
ISSN
0006-291X
DOI
10.1016/j.bbrc.2018.04.235
language
English
LU publication?
yes
id
fae7cb57-aabe-41b2-8d0d-5f0a56c6e73a
date added to LUP
2018-05-21 14:51:06
date last changed
2025-04-04 14:16:28
@article{fae7cb57-aabe-41b2-8d0d-5f0a56c6e73a,
  abstract     = {{<p>The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca<sup>2+</sup> levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.</p>}},
  author       = {{Anders, Emma and Dahl, Sara and Svensson, Daniel and Nilsson, Bengt Olof}},
  issn         = {{0006-291X}},
  keywords     = {{Antimicrobial peptide (AMP); Cathelicidin; Clathrin; Host defense peptide (HDP); Innate immunity; Osteoblast}},
  language     = {{eng}},
  month        = {{06}},
  number       = {{1}},
  pages        = {{280--285}},
  publisher    = {{Elsevier}},
  series       = {{Biochemical and Biophysical Research Communications}},
  title        = {{LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis}},
  url          = {{http://dx.doi.org/10.1016/j.bbrc.2018.04.235}},
  doi          = {{10.1016/j.bbrc.2018.04.235}},
  volume       = {{501}},
  year         = {{2018}},
}